JP6023428B2 - Composition for promoting production of laminin 332 - Google Patents

Description

  The present invention relates to a composition for promoting production of laminin 332 comprising one or more compounds selected from the group consisting of D-alanine and D-hydroxyproline, and derivatives and / or salts thereof, The present invention relates to a method for suppressing and / or ameliorating a skin disorder including an administering step.

  Laminin is a three-chain protein consisting of an α chain, a β chain, and a γ chain, and it is known that at least 15 isomers exist in a combination of 5 types of α chain, 3 types of β chain, and 3 types of γ chain. Yes. Among them, laminin 332 (α3β3γ2, laminin 5 in the previous nomenclature) is abundant in the basement membrane that separates the epidermis from the dermis, and is considered to play an important role in the structure and function of the skin (non-patent literature). 1). Laminin 332 knockout mice have a phenotype similar to that of connective epidermolysis bullosa, a human hereditary disease in which epidermis and dermis separate and form blisters, and laminin 332 is essential for adhesion between epidermis and dermis (Non-Patent Document 2). In addition, when a purified sample of laminin 332 is added to a skin equivalent model in which keratinocytes are cultured on a collagen gel in which human fibroblasts are embedded, basement membrane formation is enhanced (Non-patent Document 3). Activated proteolytic enzyme plasmin produced in epidermal cells cleaves the amino-terminal and carboxyl-terminal peptides of the α3 subunit of laminin 332 and the amino-terminal peptide of the β3 subunit. Each of the cleaved fragments includes a recognition site for a cell adhesion molecule and a binding site for type 7 collagen. Thus, laminin 332 digested with plasmin has a reduced ability to adhere to corneocytes. In addition, laminin 332 digested with plasmin has a reduced affinity with type 7 collagen. Therefore, it is considered that aging of the skin by ultraviolet irradiation or the like involves laminin 332 digestion by plasmin and a decrease in the function of the basement membrane due to this (non-patent document 4).

  Therefore, by promoting the production of laminin 332, there is a possibility that skin aging due to ultraviolet irradiation or the like can be suppressed and / or improved. Regarding factors that promote the production of laminin 332, it has recently been reported that HIF1 (Non-patent Document 5) and Smad4 (Non-patent Document 6) induce the transcription of the α3 subunit gene. However, HIF1 is a transcriptional regulator that responds to environmental stimuli such as oxygen deprivation and mechanical stimuli, and is also up-regulated by proinflammatory cytokines. Smad4 is a transcriptional regulatory factor involved in TGFβ signaling. All of these factors are proteins with large molecular weights, and their activities are regulated by modifications such as phosphorylation and the association with other subunits. Cannot be used to promote the production of laminin 332. In addition, they are regulated by a wide range of factors and are factors that greatly influence the function of the living body such as an inflammatory reaction in addition to promoting the production of laminin 332, and therefore cannot be used safely on a daily basis.

Sugawara et al., Exp Dermatol. 17 (6), 473-80 (2008) Aberdam et al., Nat. Genet. 6, 299, (1994) Amano, S.M. , SOFW J. et al. 134: 10 (2008) Amano, S.M. J. et al. Investig. Dermatol. Symp. Proc. 14: 2-7 (2009) Fitsialos, G.M. Et al. Cell Sci. 121: 2992 (2008) Zboralski, D.W. Et al., BMC Cancer, 8: 215 (2008).

  Therefore, there is a need to develop a composition that can promote the production of laminin 332 and can be used on a daily basis, and is stable and safe.

  The present invention provides a composition for promoting production of laminin 332 comprising one or more compounds selected from the group consisting of D-alanine and D-hydroxyproline, and derivatives and / or salts thereof.

  The composition for promoting production of laminin 332 of the present invention may be used for suppressing and / or improving skin conditions.

  In the composition for promoting production of laminin 332 of the present invention, the skin condition includes, but is not limited to, photoaging, wrinkles, rough skin, fine wrinkles and dryness.

  The composition for promoting production of laminin 332 of the present invention may be used as a pharmaceutical product.

  The composition for promoting production of laminin 332 of the present invention may be used as an external preparation for skin.

  The composition for promoting production of laminin 332 of the present invention may be used as a food.

  The present invention comprises the step of administering a laminin 332 production promoting composition comprising one or more compounds selected from the group consisting of D-alanine and D-hydroxyproline and derivatives and / or salts thereof. A method of suppressing and / or improving skin condition is provided.

  Skin conditions that are inhibited and / or ameliorated by the methods of the present invention include, but are not limited to, photoaging, wrinkles, rough skin, fine wrinkles and dryness.

  In the method of the present invention, the laminin 332 production promoting composition may be a pharmaceutical product.

  In the method of the present invention, the laminin 332 production promoting composition may be a skin external preparation.

  In the method of the present invention, the laminin 332 production promoting composition may be a food composition.

  In the present specification, “salts” of D-alanine and D-hydroxyproline include metal salts, amine salts and the like, provided that the promoting effect of laminin 332 production of D-alanine and D-hydroxyproline is not impaired. Any salt. The metal salt may include an alkali metal salt, an alkaline earth metal salt, and the like. The amine salt may include a triethylamine salt, a benzylamine salt, or the like.

  In the present specification, “derivatives” of D-alanine and D-hydroxyproline refer to D-alanine and D-hydroxyproline on the condition that the promoting effect of laminin 332 production of D-alanine and D-hydroxyproline is not impaired. In which the molecule is covalently bonded to any atomic group in the amino group, carboxyl group or side chain. Any one of the above atomic groups includes a protecting group such as an N-phenylacetyl group, 4,4′-dimethoxytrityl (DMT) group, and a biopolymer such as a protein, peptide, sugar, lipid, nucleic acid, and the like. Synthetic polymers such as polystyrene, polyethylene, polyvinyl, and polyester, and functional groups such as ester groups, but are not limited thereto. The ester group may include, for example, methyl ester, ethyl ester, other aliphatic esters, or aromatic esters.

  Amino acids have an L-isomer and a D-isomer as optical isomers, but natural proteins are obtained by peptide bonding of L-amino acids, and only L-amino acids are used with the exception of bacterial cell walls. Therefore, it has been considered that only L-amino acids exist in mammals including humans and only L-amino acids are used. (Tadaaki Kinouchi et al., Protein Nucleic Acid Enzyme, 50: 453-460 (2005), Reininger's Shinsei Kagaku [Up] 2nd edition pp132-147 (1993) Yodogawa Shoten, Harper Biochemistry 22nd edition pp21-30 ( 1991) Maruzen) Therefore, only L-amino acids have been used exclusively as amino acids academically and industrially.

  Exceptionally, D-amino acids are used as a starting material for antibiotics produced by bacteria, and from L-amino acids and D-amino acid mixtures obtained in equal amounts when amino acids are chemically synthesized. There is an example of a food additive in which D-amino acid is used as it is as a DL-amino acid mixture in order to save the cost of collecting only amino acids. However, there has been no example in which only D-amino acids are used industrially as a substance having physiological activity.

  Since D-serine and D-aspartic acid have a high ratio of D-form, research is relatively advanced. D-serine is localized in the cerebrum and hippocampus and has been clarified as a regulator of NMDA receptors in the brain. D-aspartic acid is localized in the testis and pineal gland and has been shown to be involved in the regulation of hormone secretion (Japanese Patent Laid-Open No. 2005-3558). However, the physiological effects of D-alanine and D-hydroxyproline in the skin have not been clarified.

  As shown in the following examples, the effect of increasing the amount of laminin 332 produced by D-alanine and D-hydroxyproline has not been known so far. Therefore, the laminin 332 production promotion composition of this invention containing D-alanine and / or D-hydroxyproline is a novel invention.

  In recent years, ddY mice were allowed to freely take a 10 mM D-amino acid aqueous solution for 2 weeks, and then the D-amino acid concentration was measured in each organ. As a result, the pineal gland showed 3-1000 pmol per gland of the pineal gland. Reported 2-500 nmol per gram wet weight (Morikawa, A. et al., Amino Acids, 32: 13-20 (2007)). Based on this, the lower limit of the daily intake of D-alanine and D-hydroxyproline contained in the composition of the present invention described below was calculated.

  D-alanine of the present invention has an effect of promoting laminin 332 production at a concentration of 0.1 μM to 1 μM for cultured human epidermal keratinocytes as shown in the following Examples. Therefore, the amount of D-alanine contained in the skin symptom improving agent, the external preparation for skin, and the food composition of the present invention is such that D-alanine in this concentration range is delivered to fibroblasts in living skin tissue. As long as the above conditions are satisfied, any content may be used. In the case where the composition of the present invention is an external preparation, the content of D-alanine may be in the range from 0.000015 wt% to 50 wt% or the maximum weight concentration that can be blended in the total composition of the present invention. . That is, when the composition is an external preparation, the content of D-alanine is preferably 0.00003 wt% to 30 wt%, and most preferably 0.0003 wt% to 3 wt%. The content of D-alanine in the case where the composition of the present invention is an internal preparation may be in the range of 0.00001 wt% to 100 wt%. When the composition of the present invention is an internal preparation, the content of D-alanine is desirably 0.00002 wt% to 80 wt%, and most desirably 0.0002 wt% to 60 wt%. In addition, the minimum of the daily intake of D-alanine contained in the composition of this invention should just be 0.01 ng per kg body weight, 0.1 ng is preferable and 1 ng is more preferable.

  The D-hydroxyproline of the present invention has an effect of promoting laminin 332 production at a concentration of 0.1 μM to 1 μM on cultured human epidermal keratinocytes as shown in the following Examples. Therefore, the amount of D-hydroxyproline contained in the skin symptom improving agent, the external preparation for skin, and the food composition of the present invention is such that D-hydroxyproline in this concentration range is delivered to fibroblasts in living skin tissue. However, any content is acceptable. When the composition of the present invention is an external preparation, the content of D-hydroxyproline is in the range from 0.000015 wt% to 50 wt% or the maximum weight concentration that can be blended in the total amount of the composition of the present invention. Good. That is, when the composition is an external preparation, the content of D-hydroxyproline is preferably 0.00003 wt% to 30 wt%, and most preferably 0.0003 wt% to 3 wt%. The content of D-hydroxyproline in the case where the composition of the present invention is an internal preparation may be in the range of 0.00001 wt% to 100 wt%. When the composition of the present invention is an internal preparation, the content of D-hydroxyproline is preferably 0.00002 wt% to 80 wt%, and most preferably 0.0002 wt% to 60 wt%. The lower limit of the daily intake of D-hydroxyproline contained in the composition of the present invention may be 0.01 ng per kg body weight, preferably 0.1 ng, and more preferably 1 ng.

  In addition to D-alanine and D-hydroxyproline, D-alanine and D-hydroxyproline salts, and / or derivatives that can release D-alanine by drug metabolizing enzymes and the like in vivo, It may further contain one or more pharmaceutically acceptable additives provided that it does not impair the laminin 332 production promoting effect of D-alanine and D-hydroxyproline. The additives include diluents and swelling agents, binders and adhesives, lubricants, glidants, plasticizers, disintegrants, carrier solvents, buffering agents, colorants, fragrances, Includes, but is not limited to, sweeteners, preservatives and stabilizers, adsorbents, and other pharmaceutical additives known to those skilled in the art.

  The composition of the present invention contains D-alanine and D-hydroxyproline, D-alanine and D-hydroxyproline salts as active ingredients, and / or D-alanine and D-hydroxy by drug metabolizing enzymes and the like in vivo. Although it is possible to prepare using only a derivative capable of releasing proline, it is usually within the range that does not impair the effects of the present invention, and is used for other skin external preparations such as cosmetics and pharmaceuticals including quasi drugs. The components can be appropriately blended as necessary. Examples of the other components (optional blending components) include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, and humectants. , Fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and the like.

  The dosage form of the composition for promoting production of laminin 332 (hereinafter referred to as “skin condition improving agent”) used for suppressing and / or improving the skin condition of the present invention is, for example, an ointment, cream, emulsion, lotion, pack, A conventional quasi-drug composition comprising an external preparation such as a gel or a patch, an oral preparation such as powder, granule, soft capsule or tablet, a nasal preparation such as nasal spray, and an injection. In addition, any material may be used as long as it is used for a pharmaceutical composition.

  The dosage form of the external preparation for skin of the present invention may be any as long as it is used for conventional external preparations for skin including ointments, creams, emulsions, lotions, packs, gels, patches and the like.

  The food composition of the present invention releases D-alanine and D-hydroxyproline by D-alanine and D-hydroxyproline, D-alanine and D-hydroxyproline salts, and / or drug metabolizing enzymes and the like in vivo. In addition to the derivatives that can be used, it may contain seasonings, coloring agents, preservatives, and other food-acceptable ingredients provided that they do not impair the promoting effect of D-alanine and D-hydroxyproline on laminin 332 production. .

  The food composition of the present invention is used in conventional food compositions such as candy, cookies, miso, French dressing, mayonnaise, French bread, soy sauce, yogurt, sprinkle, seasoning / natto sauce, natto, moromi black vinegar, etc. Any material can be used, and the present invention is not limited to the above examples.

The graph which shows the influence of D-alanine with respect to a KC cell. The graph which shows the effect of D-alanine with respect to the production of laminin 332 in a KC cell. The graph which shows the influence of D-alanine with respect to a HaCaT cell. The graph which shows the influence of D-hydroxyproline with respect to a HaCaT cell. Graph showing the effect of D-alanine on laminin 332 production in HaCaT cells. The graph which shows the effect of D-hydroxyproline with respect to the production of laminin 332 in a HaCaT cell. Graph showing the effect of various concentrations of D-alanine on laminin 332 production in HaCaT cells. Graph showing the effect of various concentrations of D-hydroxyproline on laminin 332 production in HaCaT cells. Graph showing the effect of various concentrations of D-aspartic acid on laminin 332 production in HaCaT cells. Graph showing the effect of various concentrations of D-asparagine on laminin 332 production in HaCaT cells. Graph showing the effect of various concentrations of D-proline on laminin 332 production in HaCaT cells. Graph showing the effect of various concentrations of D-serine on laminin 332 production in HaCaT cells.

  The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims.

  All documents mentioned herein are hereby incorporated by reference in their entirety.

1. 1. Promotion of laminin 332 production by addition of alanine and hydroxyproline 1-1. Materials and Methods (1) Cells Cells include HaCaT cells derived from human epidermal cells (H. Hans et al., Experimental Cell Research 239: 399 (1998)) and KC cells derived from human keratinocytes (Sanko Junyaku Co., Ltd.) , Manufacturer: LONZA Walkersville Inc.). The cells are seeded at a density of ⁴ × 10 ⁴ per well in a 24-well plate, and a medium in which 0.1% BSA is added to a cell culture medium (D-MEM (1 g / L glucose), Wako Pure Chemical Industries, Ltd.) (Hereinafter, referred to as “normal medium”) was cultured at 37 ° C., 5% CO ₂ and saturated water vapor atmosphere for 24 hours.

(2) Addition of alanine and hydroxyproline Thereafter, the cells are either 1 μM of L- or D-alanine, 0.5 μM of both L- and D-alanine, 1 μM of L- or D-hydroxyproline, Both L- and D-hydroxyproline at 0.5 μM were switched to the medium added to the normal medium and cultured for 24 hours. The normal medium without the addition of alanine and hydroxyproline was used as a negative control. The D-hydroxyproline used in this example was 4-cis-D-hydroxyproline.

(3) Quantification of laminin 332 production amount After completion of the culture, the medium was collected and centrifuged at 3000 rpm for 5 minutes, and the concentration of laminin 332 in the supernatant was determined by ELISA (Amano, S. et al., J. Immunol. Methods, 224: 161 (1999)). The ELISA method is a sandwich ELISA method using BM165, which is a monoclonal antibody against the α3 chain of laminin 5, and a biotinylated conjugate of 6F12, which is a monoclonal antibody against the β3 chain of laminin 5. Horseradish peroxidase-labeled avidin D ( Detected by Vector Labs, catalog number A-2004). PBS was used as a control.

(4) Quantification of the number of viable cells After recovering the medium, the cells were washed with PBS, and alamarBlue (trademark, Biosource, Biosource International) was added to a final concentration of 10%. Two hours later, Ahmed S. A. (J. Immunol. Method. 170, 211-224 (1994)) and the manufacturer's instructions, the fluorescence intensity of the alamarBlue solution was measured at an excitation wavelength of 544 nm and a fluorescence wavelength of 590 nm.

1-2. Results (1) Quantification of KC Viable Cell Count FIG. 1 shows the experimental results of examining the influence of alanine addition on the proliferation of KC cells. The error bar for each experimental condition indicates the standard deviation of the measured value of the experimental result repeated three times under the same condition.

  In KC cells, the relative value of the fluorescence intensity of the negative control alamarBlue ™ (hereinafter the same) was 50. The fluorescence intensity of KC cells cultured in 1 μM L-alanine supplemented medium, 1 μM D-alanine supplemented medium, and 0.5 μM each of L- and D-alanine supplemented medium was 40, 45 and 50, respectively. Met. In KC cells, the fluorescence intensity of alamarBlue ™ in cells cultured in alanine supplemented media was not significantly different in the Tukey-Kramer test compared to the negative control. Therefore, it was shown that L- and D-alanine are not cytotoxic to KC cells.

(2) Production of laminin 332 by KC cells Fig. 2 shows the results of experiments examining the effect of alanine addition on laminin 332 production by KC cells. The vertical axis of the graph of FIG. 2 is a quotient obtained by dividing the absorbance of ELISA measurement proportional to the laminin 332 concentration in the culture supernatant of each well by the fluorescence intensity of alamarBlue (trademark) proportional to the number of cells in each well (hereinafter referred to as the quotient). , “Relative value of laminin 332 concentration per number of cells”). The error bar for each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated three times under the same condition. An asterisk (**) indicates that p is less than 1% in the Tukey-Kramer test.

  The relative value of laminin 332 concentration per cell number was 0.35 in the negative control. The relative value of laminin 332 concentration per cell number of KC cells cultured in 1 μM L-alanine supplemented medium, 1 μM D-alanine supplemented medium, and 0.5 μM each of L- and D-alanine supplemented medium is , 0.40, 0.50 and 0.45, respectively. There was a significant difference between the addition of 1 μM D-alanine and the negative control in the Tukey-Kramer test with p of less than 1%. Thus, the promotion of laminin 332 production in KC cells by adding D-alanine was proved.

(3) Quantification of the number of live cells of HaCaT FIG. 3 and FIG. 4 show the experimental results of examining the effects of addition of alanine and hydroxyproline on the growth of HaCaT cells. Hereinafter, D-hydroxyproline (D-Hyp) refers to 4-cis-D-hydroxyproline. The error bar for each experimental condition indicates the standard deviation of the measured value of the experimental result repeated three times under the same condition.

  In HaCaT cells, the fluorescence intensity of the negative control alamarBlue ™ was 300. The fluorescence intensity of HaCaT cells cultured in 1 μM L-alanine-added medium, 1 μM D-alanine-added medium, and 0.5 μM each of L- and D-alanine-added medium was 250 (see FIG. 3). The fluorescence intensity of HaCaT cells cultured in 1 μM L-hydroxyproline supplemented medium, 1 μM D-hydroxyproline supplemented medium, and 0.5 μM each of L- and D-hydroxyproline supplemented medium was 280. (FIG. 4). Even in HaCaT cells, the fluorescence intensity of alamarBlue ™ in cells cultured in a medium supplemented with alanine and D-hydroxyproline was not significantly different in the Tukey-Kramer test compared to the negative control. Therefore, it was shown that L- and D-alanine and L- and D-hydroxyproline are not cytotoxic to HaCaT cells.

(4) Laminin 332 production of HaCaT cells by addition of alanine FIG. 5 shows the experimental results of examining the effect of alanine addition on laminin 332 production in HaCaT cells. The vertical axis of the graph of FIG. 5 shows the relative value of laminin 332 concentration per number of cells in the culture supernatant of each well. The error bar for each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated three times under the same condition. An asterisk (**) indicates that p is less than 1% in the Scheffe's F-test, and an asterisk (*) indicates that p is less than 5%.

  The relative value of laminin 332 concentration per cell number was 0.04 in the negative control. The relative value of the laminin 332 concentration per number of HaCaT cells cultured in 1 μM L-alanine supplemented medium, 1 μM D-alanine supplemented medium, and 0.5 μM each of L- and D-alanine supplemented medium is , 0.07, 0.11 and 0.10, respectively. In the Scheffe's F-test, p was less than 1% between the addition of 1 μM D-alanine and the negative control, and between the addition of 0.5 μM D-alanine and L-alanine and the negative control, Scheffe's. p was less than 5% in the s F-test, and p was less than 5% in the Scheffe's F-test between 1 μM D-alanine and 1 μM L-alanine, both of which were significantly different. Thus, similarly to KC cells, the promotion of laminin 332 production by addition of D-alanine has been demonstrated in HaCaT cells.

(5) Production of Laminin 332 in HaCaT Cells by Addition of Hydroxyproline FIG. 6 shows the experimental results of examining the effect of hydroxyproline addition on the production of laminin 332 in HaCaT cells. The vertical axis of the graph of FIG. 6 shows the relative value of laminin 332 concentration per number of cells in the culture supernatant of each well. The error bar for each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated three times under the same condition.

  The relative value of laminin 332 concentration per cell number was 0.04 in the negative control. Laminin 332 concentration per cell number of HaCaT cells cultured in 1 μM L-hydroxyproline supplemented medium, 1 μM D-hydroxyproline supplemented medium, and 0.5 μM each of L- and D-hydroxyproline supplemented medium. The relative values were 0.05, 0.065 and 0.06, respectively.

2. 2. Comparison of laminin 332 production by adding various amino acids 2-1. Materials and Methods HaCaT cells were used as the cells and cultured in the same manner as in Example 1. The cells are then 10 nM, 100 nM and 1000 nM L- or D-alanine, L- or D-hydroxyproline, L- or D-aspartic acid, L- or D-asparagine, L- or D -Proline, L- or D-serine amino acid was added to the normal medium and cultured for 24 hours. The normal medium without the amino acid was used as a negative control. The D-hydroxyproline used in this example was 4-cis-D-hydroxyproline. The production amount of laminin 332 was measured in the same manner as in Example 1. In the following experiments, no cytotoxicity due to the addition of amino acids was observed at any concentration, so the amount of laminin 332 produced by the cells was compared under each experimental condition.

2-2. Results (1) Addition of alanine FIG. 7 shows the experimental results of examining the effects of adding various concentrations of alanine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 7 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.7 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-alanine and D-alanine supplemented media were 1.7 ng / mL and 1.7 ng / mL, 2.3 ng / mL and 3.4 ng / mL, 2.8 ng / mL and 4.8 ng / mL. From the above results, it was shown that D-alanine promotes laminin 332 production at a concentration of 100 ng / mL or more.

(2) Hydroxyproline addition FIG. 8 shows the experimental results of examining the effects of various concentrations of hydroxyproline on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 8 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.5 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-hydroxyproline and D-hydroxyproline supplemented media were 1.5 ng / mL and 1.5 ng / mL and 2.3 ng / mL, respectively. mL and 3.3 ng / mL and 2.7 ng / mL and 4.4 ng / mL. From the above results, it was shown that D-hydroxyproline promotes laminin 332 production at a concentration of 100 ng / mL or more.

(3) Aspartic Acid Addition FIG. 9 shows the experimental results of examining the effects of various concentrations of aspartic acid on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 9 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.7 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-aspartic acid-added medium and D-aspartic acid-added medium were 1.8 ng / mL and 1.9 ng / mL and 1.8 ng / mL, respectively. mL and 1.9 ng / mL, and 2.0 ng / mL and 1.7 ng / mL. From the above results, it was shown that L- and D-aspartic acid did not promote laminin 332 production.

(4) Addition of asparagine FIG. 10 shows the experimental results of examining the effects of adding various concentrations of asparagine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 10 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.5 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-asparagine and D-asparagine supplemented media were 1.6 ng / mL and 1.5 ng / mL, 1.5 ng / mL and 1.5 ng / mL, 1.6 ng / mL and 1.5 ng / mL. From the above results, it was shown that L- and D-asparagine do not promote laminin 332 production.

(5) Addition of proline FIG. 11 shows the experimental results of examining the effect of addition of various concentrations of proline on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 11 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.8 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-proline and D-proline supplemented media were 1.8 ng / mL and 2.0 ng / mL, 1.9 ng / mL and 1.9 ng / mL, 1.9 ng / mL and 1.9 ng / mL. From the above results, it was shown that L- and D-proline do not promote laminin 332 production.

(6) Addition of Serine FIG. 12 shows the experimental results of examining the effect of adding various concentrations of serine on the production of laminin 332 in HaCaT cells. The vertical axis of the graph in FIG. 12 indicates the laminin 332 concentration (ng / mL) in the culture supernatant of each well. The error bar of each experimental condition indicates the standard deviation of the calculated value of the experimental result repeated four times under the same condition.

  The laminin 332 concentration was 1.7 ng / mL in the negative control. The laminin 332 concentrations of HaCaT cells cultured in 10 nM, 100 nM and 1000 nM L-serine and D-serine supplemented media were 1.8 ng / mL and 1.8 ng / mL, 1.9 ng / mL and 1.8 ng / mL, 1.9 ng / mL and 1.8 ng / mL. From the above results, it was shown that L- and D-serine do not promote laminin 332 production.

Conclusion From the experimental results of Examples 1 and 2, laminin 332 production promoting effect was observed with D-alanine and D-hydroxyproline, but not with aspartic acid, asparagine, proline and serine. Thus, it was suggested that D-alanine and D-hydroxyproline can suppress and / or improve the skin condition by promoting the production of laminin 332, which plays an important role in the structure and function of the basement membrane.

  In accordance with the present invention, emulsion preparation, patch, tablet, soft capsule, granule, drink, candy, cookie, miso, French dressing, mayonnaise, French bread, soy sauce, yogurt, containing D-alanine and / or D-hydroxyproline, Formulation examples of sprinkles, seasoning / natto sauce, natto, moromi black vinegar, cream, body cream, gel agent, peel-off mask, impregnation mask, emulsion, lotion and aerosol are shown below. These formulation examples are listed for the purpose of illustration and are not intended to limit the technical scope of the present invention.

Formulation Example 1 (Emulsion formulation)
(Composition) Compounding amount (% by weight)
D-alanine or D-hydroxyproline 0.42
Behenyl alcohol 0.2
Cetanol 0.5
Glycerin mono fatty acid ester 1.8
Hardened castor oil POE (60) 1.0
White petrolatum 2.0
Liquid paraffin 10.0
Isopropyl myristate 3.0
Methyl polysiloxane (6cs) 1.5
Concentrated glycerin 13.0
Dipropylene glycol 2.0
Carboxyvinyl polymer 0.25
Sodium hyaluronate 0.005
Potassium hydroxide Appropriate amount of lactic acid Appropriate amount of sodium edetate Appropriate amount of ethyl paraben Appropriate amount of purified water Residue

100.000

Formulation Example 2 (Patch)
(Composition) Compounding amount (% by weight)
D-alanine or D-hydroxyproline 0.3
Polyacrylic acid 3.0
Sodium polyacrylate 2.5
Gelatin 0.5
Sodium carboxymethylcellulose 4.0
Polyvinyl alcohol 0.3
Concentrated glycerin 14.0
1,3-butylene glycol 12.0
Aluminum hydroxide 0.1
Sodium edetate 0.03
Methylparaben 0.1
Purified water residue

100.00

Formulation Example 3 (tablet)
(Composition) Formulation amount (mg / tablet)
D-alanine or D-hydroxyproline 360.5
Lactose 102.4
Carboxymethylcellulose calcium 29.9
Hydroxypropylcellulose 6.8
Magnesium stearate 5.2
Crystalline cellulose 10.2

515.0

Formulation Example 4 (tablet)
(Composition) Formulation amount (mg / tablet)
Sucrose ester 70
Crystalline cellulose 74
Methylcellulose 36
Glycerin 25
D-alanine or D-hydroxyproline 475
N-acetylglucosamine 200
Hyaluronic acid 150
Vitamin E 30
Vitamin B6 20
Vitamin B2 10
α-Lipoic acid 20
Coenzyme Q10 40
Ceramide (konjac extract) 50
L-proline 300

1500

Formulation Example 5 (soft capsule)
(Composition) Blending amount (mg / 1 capsule)
Edible soybean oil 530
Eucommia extract 50
Carrot extract 50
D-alanine or D-hydroxyproline 100
Royal Jelly 50
Maca 30
GABA 30
Beeslow 60
Gelatin 375
Glycerin 120
Glycerin fatty acid ester 105

1500

Formulation Example 6 (soft capsule)
(Composition) Blending amount (mg / 1 capsule)
Brown rice germ oil 659
D-alanine or D-hydroxyproline 500
Resveratrol 1
Lotus germ extract 100
Elastin 180
DNA 30
Folic acid 30

1500

Formulation Example 7 (granule)
(Composition) Blending amount (mg / pack)
D-alanine or D-hydroxyproline 400
Vitamin C 100
Soy isoflavone 250
Reduced lactose 300
Soybean oligosaccharide 36
Erythritol 36
Dextrin 30
Fragrance 24
Citric acid 24

1200

Formulation Example 8 (Drink)
(Composition) Compounding amount (in g / 60 mL)
Eucommia extract 1.6
Carrot extract 1.6
D-alanine or D-hydroxyproline 1.6
Reduced maltose starch syrup 28
Erythritol 8
Citric acid 2
Fragrance 1.3
N-acetylglucosamine 1
Hyaluronic acid Na 0.5
Vitamin E 0.3
Vitamin B6 0.2
Vitamin B2 0.1
α-Lipoic acid 0.2
Coenzyme Q10 1.2
Ceramide (konjac extract) 0.4
L-proline 2
Purified water residue

60

Formulation Example 9 (candy)
(Composition) Compounding amount (% by weight)
Sugar 50
Minamata 48
D-alanine or D-hydroxyproline 1
Fragrance 1

100

Formulation Example 10 (Cookie)
(Composition) Compounding amount (% by weight)
Soft flour 45.0
Butter 17.5
Granulated sugar 20.0
D-alanine or D-hydroxyproline 4.0
Egg 12.5
Fragrance 1.0

100.0

Production Method of Formulation Example 10 (Cookie) Granulated sugar was gradually added while stirring butter, and eggs and flavors and D-alanine or D-hydroxyproline were added and stirred. After thorough mixing, a weak flour that was shaken uniformly was added and stirred at a low speed. Then, it shape | molded and baked for 170 degreeC15 minutes, and was set as the cookie.

Formulation Example 11 (Miso)
(Composition) Blending amount (g)
Soybean 1000
Rice bran 1000
Salt 420
D-alanine or D-hydroxyproline 158
Water residue

4000

Production method of Formulation Example 11 (Miso) Rice koji and salt are mixed well. After soaking the washed soybeans in 3 times the amount of water overnight, drain the water, boil it with fresh water, and pour it into the sardine. Boiled broth (seed water) is collected, and D-alanine or D-hydroxyproline is dissolved to 10% w / v. Grind the boiled beans immediately, add rice bran mixed with salt, and mix evenly until adding the seed water in which the above D-alanine or D-hydroxyproline is dissolved until it is as hard as clay. Pack the rolled-up dumplings into the corners without any gaps, flatten the surface, cover with wraps and seal. After 3 months, transfer the container, flatten the surface and cover with wrap. In addition, you may use the rice bran which produces many D-alanine or D-hydroxyproline instead of adding D-alanine or D-hydroxyproline to seed water. In order to obtain the rice bran, it can be selected by quantifying D-alanine or D-hydroxyproline by the method described in JP-A-2008-185558. Moreover, you may add D-alanine, D-hydroxyproline, or those salts to commercially available miso.

Formulation Example 12 (French dressing)
(Composition) Blending amount (g)
Salad oil 27.0
Vinegar 30.0
Sodium chloride 0.9
D-alanine or D-hydroxyproline 1.1
Pepper 1.0

60.0

Production Method of Formulation Example 12 (French Dressing) After adding sodium chloride and D-alanine or D-hydroxyproline to vinegar, dissolve well by stirring. Add salad oil, stir well and add pepper.

Formulation Example 13 (mayonnaise)
(Composition) Blending amount (g)
Salad oil 134.0
Vinegar 5
Sodium chloride 0.9
D-alanine or D-hydroxyproline 1
Yolk 18
Sugar 0.2
Pepper 0.9

160.0

Production method of Formulation Example 13 (mayonnaise) Vinegar, sodium chloride and pepper, and D-alanine or D-hydroxyproline are added to egg yolk (room temperature) and sufficiently stirred with a whisk. Stirring is continued while adding salad oil little by little to make an emulsion. Finally add sugar and stir.

Formulation Example 14 (French bread)
(Composition) Blending amount (g)
Powerful powder 140
Soft flour 60
Sodium chloride 3
Sugar 6
D-alanine or D-hydroxyproline 2
Dry yeast 4
Lukewarm water 128

343

Production method of Formulation Example 14 (French bread) Pre-fermented with 1 g of sugar and dry yeast in lukewarm water. Intense flour, weak flour, sodium chloride, 5 g sugar and D-alanine or D-hydroxyproline are placed in a bowl and prefermented yeast is placed therein. After kneading sufficiently, it is made into a sphere and subjected to primary fermentation at 30 ° C. The dough is kneaded again, rested, shaped into a suitable shape, and finally fermented using an electronic fermenter. Bake in a 220 ° C oven for 30 minutes.

Formulation Example 15 (soy sauce)
(Composition) Blending amount (g)
Commercial soy sauce 900
D-alanine or D-hydroxyproline 100

1000

Production method of Formulation Example 15 (soy sauce) D-alanine or D-hydroxyproline or a salt thereof is added to commercially available soy sauce and well stirred. Further, instead of adding D-alanine or D-hydroxyproline or a salt thereof, soy sauce may be brewed using koji that produces a large amount of D-alanine or D-hydroxyproline. In order to obtain the said wrinkles, it can select by quantifying D-alanine or D-hydroxyproline by the method of Unexamined-Japanese-Patent No. 2008-185558.

Formulation Example 16 (yogurt)
(Composition) Blending amount (g)
Milk 880
L. Bulgaricus 50
S. Thermophilus 50
D-alanine or D-hydroxyproline 20

1000

Production method of Formulation Example 16 (yogurt) Fermentation is performed at 40 ° C to 45 ° C. Other commercially available inoculums may be used, and D-alanine or D-hydroxyproline or a salt thereof may be added to commercially available yogurt. Moreover, you may use the microbe which produces many D-alanine or D-hydroxyproline instead of adding D-alanine or D-hydroxyproline, or those salts. In order to obtain the said microbe, it can select by quantifying D-alanine or D-hydroxyproline by the method of Unexamined-Japanese-Patent No. 2008-185558.

Formulation Example 17 (sprinkle)
(Composition) Blending amount (g)
D-alanine or D-hydroxyproline 50
Paste 15
L-glutamic acid Na 10
Sodium chloride 2
Roasted sesame 10
Crusher Clause 10
Sugar 1
Soy sauce 2

100

Formulation Example 18 (Seasoning and natto sauce)
(Composition) Blending amount (g)
Commercial natto sauce 9
D-alanine or D-hydroxyproline 1

10

Formulation Example 19 (Natto)
(Composition) Blending amount (g)
Commercial natto 19.9
D-alanine or D-hydroxyproline 0.1

20

Production method of Formulation Example 19 (Natto) D-alanine or D-hydroxyproline or a salt thereof is added to commercially available natto and stirred well. Moreover, you may make natto using the microbe which produces many D-alanine or D-hydroxyproline instead of adding D-alanine or D-hydroxyproline, or those salts. In order to obtain the said microbe, it can select by quantifying D-alanine or D-hydroxyproline by the method of Unexamined-Japanese-Patent No. 2008-185558.

Formulation Example 20 (Moromi black vinegar)
(Composition) Blending amount (g)
Commercially available moromi black vinegar 900
D-alanine or D-hydroxyproline 100

1000

Production Method of Formulation Example 20 (Moromi Black Vinegar) D-alanine or D-hydroxyproline or a salt thereof is added to commercially available moromi black vinegar and stirred well. Instead of adding D-alanine, D-hydroxyproline, or salts thereof, vinegar, black vinegar, and moromi may be made using bacteria that produce a large amount of D-alanine or D-hydroxyproline. In order to obtain the said microbe, it can select by quantifying D-alanine or D-hydroxyproline by the method of Unexamined-Japanese-Patent No. 2008-185558.

Formulation Example 21 (cream)
(Composition) Compounding amount (% by weight)
Liquid paraffin 3
Vaseline 1
Dimethylpolysiloxane 1
Stearyl alcohol 1.8
Behenyl alcohol 1.6
Glycerin 8
Dipropylene glycol 5
Macadamia nut oil 2
Hardened oil 3
Squalane 6
Stearic acid 2
Cholesteryl hydroxystearate 0.5
Cetyl 2-ethylhexanoate 4
Polyoxyethylene hydrogenated castor oil 0.5
Self-emulsifying glyceryl monostearate 3
Potassium hydroxide 0.15
Sodium hexametaphosphate 0.05
Trimethylglycine 2
α-tocopherol 2-L-ascorbic acid
Potassium phosphate diester 1
Tocopherol acetate 0.1
D-alanine or D-hydroxyproline 4
Paraben appropriate amount edetate trisodium 0.05
4-t-butyl-4′-methoxydibenzoylmethane 0.05
Diparamethoxycinnamic acid mono-2-ethylhexanoate glyceryl 0.05
Colorant Appropriate amount Carboxyvinyl polymer 0.05
Purified water residue

100.00

Formulation Example 22 (Body Cream)
(Composition) Compounding amount (% by weight)
Dimethylpolysiloxane 3
Decamethylcyclopentasiloxane 13
Dodecamethylcyclohexasiloxane 12
Polyoxyethylene methylpolysiloxane copolymer 1
Ethanol 2
Isopropanol 1
Glycerin 3
Dipropylene glycol 5
Polyethylene glycol 6000 5
Sodium hexametaphosphate 0.05
Tocopherol acetate 0.1
D-alanine or D-hydroxyproline 5
Fennel extract 0.1
Hamelis extract 0.1
Carrot extract 0.1
L-menthol appropriate amount paraoxybenzoate appropriate amount edetate trisodium 0.05
Dimorpholinopyridazinone 0.01
Methyl bis (trimethylsiloxy) trimethoxycinnamate
Silyl isopentyl 0.1
Yellow iron oxide Appropriate amount Cobalt titanate Appropriate amount Dimethyl distearyl ammonium hectorite 1.5
Polyvinyl alcohol 0.1
Hydroxyethyl cellulose 0.1
Trimethylsiloxysilicic acid 2
Perfume Appropriate amount of purified water

100.00

Formulation Example 23 (gel agent)
(Composition) Compounding amount (% by weight)
Dimethylpolysiloxane 5
Glycerin 2
1,3-butylene glycol 5
Polyethylene glycol 1500 3
Polyethylene glycol 20000 3
Cetyl octanoate 3
Citric acid 0.01
Sodium citrate 0.1
Sodium hexametaphosphate 0.1
Dipotassium glycyrrhizinate 0.1
D-alanine or D-hydroxyproline 2
Tocopherol acetate 0.1
Ogon Extract 0.1
Yukinoshita extract 0.1
Edetate trisodium 0.1
Xanthan gum 0.3
Acrylic acid / methacrylic acid
Alkyl copolymer (Pemulene TR-2) 0.05
Agar powder 1.5
Phenoxyethanol Appropriate amount Dibutylhydroxytoluene Appropriate amount of purified water Residue

100.00

Formulation Example 24 (Peel-off mask)
(Composition) Compounding amount (% by weight)
Ethanol 10
1,3-butylene glycol 6
Polyethylene glycol 4000 2
Olive oil 1
Macadamia nut oil 1
Phytosteryl hydroxystearate 0.05
Lactic acid 0.05
Sodium lactate 0.1
L-ascorbic acid sulfate disodium 0.1
α-tocopherol 2-L-ascorbic acid
Potassium phosphate diester 0.1
D-alanine or D-hydroxyproline 10
Fish collagen 0.1
Chondroitin thorium sulfate 0.1
Sodium carboxymethylcellulose 0.2
Polyvinyl alcohol 12
Paraoxybenzoic acid ester Appropriate amount of fragrance Appropriate amount of purified water Residue

100.00

Formulation Example 25 (impregnation mask)
(Composition) Compounding amount (% by weight)
Glycerin 1
1,3-butylene glycol 8
Xylit 2
Polyethylene glycol 1500 2
Rosemary oil 0.01
Sage oil 0.1
Citric acid 0.02
Sodium citrate 0.08
Sodium hexametaphosphate 0.01
Hydroxypropyl-β-cyclodextrin 0.1
D-alanine or D-hydroxyproline 0.5
Birch extract 0.1
Lavender oil 0.01
Xanthan gum 0.05
Carboxyvinyl polymer 0.15
P-Hydroxybenzoate appropriate amount purified water remaining

100.00

Formulation Example 26 (Emulsion)
(Composition) Compounding amount (% by weight)
Liquid paraffin 7
Vaseline 3
Decamethylcyclopentasiloxane 2
Behenyl alcohol 1.5
Glycerin 5
Dipropylene glycol 7
Polyethylene glycol 1500 2
Jojoba oil 1
Isostearic acid 0.5
Stearic acid 0.5
Behenic acid 0.5
Tetra-2-ethylhexanoic acid pentaerythrit 3
Cetyl 2-ethylhexanoate 3
Glycerol monostearate 1
Polyoxyethylene glyceryl monostearate 1
Potassium hydroxide 0.1
Sodium hexametaphosphate 0.05
Stearyl glycyrrhetinate 0.05
D-alanine or D-hydroxyproline 1
Royal Jelly Extract 0.1
Yeast extract 0.1
Tocopherol acetate 0.1
Acetylated sodium hyaluronate 0.1
Edetate trisodium 0.05
4-t-butyl-4'-methoxydibenzoylmethane 0.1
2-Ethylhexyl paramethoxycinnamate 0.1
Carboxyvinyl polymer 0.15
Paraben Appropriate amount of perfume Appropriate amount of purified water Residual

100.00

Formulation Example 27 (milky lotion)
(Composition) Compounding amount (% by weight)
Dimethylpolysiloxane 2
Behenyl alcohol 1
Batyl alcohol 0.5
Glycerin 5
1,3-butylene glycol 7
Erythritol 2
Hardened oil 3
Squalane 6
Tetra-2-ethylhexanoic acid pentaerythlit 2
Polyoxyethylene glyceryl isostearate 1
Polyoxyethylene glyceryl monostearate 1
D-alanine or D-hydroxyproline 0.3
Potassium hydroxide appropriate amount Sodium hexametaphosphate 0.05
Phenoxyethanol appropriate amount carboxyvinyl polymer 0.1
Purified water residue

100.00

Formulation Example 28 (lotion)
(Composition) Compounding amount (% by weight)
Ethyl alcohol 5
Glycerin 1
1,3-butylene glycol 5
Polyoxyethylene polyoxypropylene
Decyl tetradecyl ether 0.2
Sodium hexametaphosphate 0.03
Trimethylglycine 1
Sodium polyaspartate 0.1
α-tocopherol 2-L-ascorbic acid
Potassium phosphate diester 0.1
Thiotaurine 0.1
D-alanine or D-hydroxyproline 8
EDTA3 sodium 0.1
Carboxyvinyl polymer 0.05
Potassium hydroxide 0.02
Phenoxyethanol appropriate amount perfume appropriate amount purified water remaining

100.00

Formulation Example 29 (lotion)
(Composition) Compounding amount (% by weight)
Ethanol 10
Dipropylene glycol 1
Polyethylene glycol 1000 1
Polyoxyethylene methyl glucoside 1
Jojoba oil 0.01
Glyceryl tri-2-ethylhexanoate 0.1
Polyoxyethylene hydrogenated castor oil 0.2
Polyglyceryl diisostearate 0.15
N-stearoyl-L-glutamate sodium 0.1
Citric acid 0.05
Sodium citrate 0.2
Potassium hydroxide 0.4
Dipotassium glycyrrhizinate 0.1
Arginine hydrochloride 0.1
L-ascorbic acid 2-glucoside 2
D-alanine or D-hydroxyproline 0.5
Edetate trisodium 0.05
2-Ethylhexyl paramethoxycinnamate 0.01
Dibutylhydroxytoluene Appropriate amount Paraben Appropriate amount Deep sea water 3
Perfume Appropriate amount of purified water

100.00

Formulation Example 30 (Aerosol urea external preparation stock solution)
(Composition) Compounding amount (% by weight)
Ethanol 15.0
Polyoxyethylene hydrogenated castor oil 50 1.5
Diphenhydramine 1.0
Jibcaine 2.0
Tocopherol acetate 0.5
D-alanine or D-hydroxyproline 0.1
Isostearic acid 0.1
1,3-butylene glycol 3.0
Polyethylene glycol 400 3.0
Camphor 0.05
Urea 20.0
Purified water residue

100.00

Formulation Example 31 (Aerosol urea propellant)
(Composition) Compounding amount (% by weight)
Aerosol urea external preparation stock solution 65.0
Dimethyl ether 35.0

100.00

Formulation Example 31 (Aerosol Urea Propellant) Filling Method An aerosol urea preparation is prepared by filling an inner surface Teflon (registered trademark) coated pressure-resistant aerosol aluminum can with an aerosol urea external preparation stock solution and dimethyl ether.

Claims (5)

D- alanine or comprising a salt thereof, 332 production accelerating composition laminin in the skin tissues (except skin roughening improvement applications). Characterized in that it is used to suppress and / or improving the skin conditions, except for rough skin composition of claim 1. It said skin condition is photoaging, wrinkling, characterized in that it is selected from the group consisting of fine lines and dry composition according to claim 2.   The composition according to any one of claims 1 to 3, wherein the composition is used as a medicine.   The composition according to any one of claims 1 to 3, wherein the composition is used as an external preparation for skin.

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